Parainfluenza virus vaccine for the control of canine tracheo-bronchitis (kennel cough)

ABSTRACT

A kennel cough vaccine, prepared by passaging an SV5-like parainfluenza virus in Madin Darby Canine Kidney cells, is described as being effective in inducing immunity to canine tracheobronchitis (kennel cough).

United States Patent [1 1 Lief [4 1 Sept. 9, 1975 PARAINFLUENZA VIRUS VACCINE FOR THE CONTROL OF CANINE TRACHEO-BRONCHITIS (KENNEL COUGH) [75] Inventor: Florence S. Lief, Bala Cynwyd, Pa.

[73] Assignee: The Trustees of the University of Pennsylvania, Philadelphia, Pa.

[22] Filed: Dec. 18, 1974 [21] Appl. No.: 533,794

Related U.S. Application Data [63] Continuation-in-part of Ser. No. 440,015, Feb. 6,

i974, abandoned.

[52] U.S. Cl. 424/89; l95/l.l; 195/13; l95/l.8 [51] Int. Cl? C12K 7/00 [58] Field of Search 424/89; 195/l.1-l.8

[56] References Cited OTHER PUBLlCATIONS Wright et al., J. Small Anim. Pract., l5( 1 2735 Jan. 1974, Canine Respiratory Virus Infections. OBrien et al., Amer. Anim. Hosp. Ass. Proc., 38: 178-179 (l97l), Current Concepts in Respiratory Disease Dogs. Goodwin et al., Curr. Vet. Therap., 4: 643-644 (1971), Upper Respiratory Viral Diseases of the Dog.

Binn et al., Amer. J. Vet. Res., 31(4): 697-702 Apr. 1970, Viral Antibody Patterns in Laboratory Dogs with Respiratory Disease.

Tribe et al., J. Small Anim. Pract., 14(5): 251-255 May 1973, Protection of Dogs Against Canine Hepatitis with Toronto A26/6l Virus (Kennel Cough [solate) Hexon Antigen.

Appel'et al., Amer. Vet. Med, Ass. J., 156(l2, pt.l): l778-l781, June 15, 1970, SV-S-Like Parainfluenza Virus in Dogs."

Binn et al., Amer. Vet. Med. Ass. 1., l56(l2,Pt. l): l774l777, June 15, 1970, Comments on Epizootiology of Parainfluenza SV-5 in Dogs."

Bittle et al., Amer. Vet. Med. Ass. J., 156(12, Pt.l): 17714773, June 15, 1970, The Epizootiology of Canine Parainfluenza."

Primary Examiner-Shep K. Rose Attorney, Agent, or FirmPaul & Paul 5 7 ABSTRACT 25 Claims, N0 Drawings PARAINFLUENZA VIRUS VACCINE FOR THE CONTROL OF CANINE TRACHEO-BRONCI-IITIS (KENNEL COUGH) RELATED CASE This application is a continuation-in-part of my co pending application Ser. No. 440,015, filed Feb. 6. 1974, now abandoned.

This invention relates to the manufacture and administration of a parainfluenza live-virus vaccine which is capable of inducing immunity to canine tracheobronchitis (commonly known as kennel cough).

BACKGROUND OF THE INVENTION Respiratory disease among closed populations of dogs assembled in kennels and other similar facilities is quite common. In one dog population, for example, respiratory infections accounted for the deaths of more than half of those dogs admitted for a six week stay in the facility. While several viruses have been implicated as the cause of the disease, recently a single SV -,-like parainfluenza virus has been isolated from diseased dogs throughout the United States. Table I gives some indication of the nature of populations and extent of geographical dissemination of this virus. Tables II and Ill show that Sv -like canine parainfluenza isolates from distant origins are antigenically identical. Although other viruses such as canine distemper, ICH and the related adenovirus, A/Toronto 26 and occasionally a herpes virus. are capable of producing respiratory dis- Location ease signs which are clinically difficult to differentiate from the parainfluenza virus induced disease. the evidence suggests that this parainfluenza virus is a significant cause of kennel cough. particularly since ICH and canine distemper vaccines are available.

Research has indicated that this SV -,-like parainfluenza virus can establish infection in virtually 100 /1 of dogs without preexisting antibody, that once infected.

the disease can be transmitted by contact to other antil0 body-free dogs in series, and that recovered dogs challenged by the same virus two and one half months later are resistant to reinfection. These results are substantiated by field studies which indicate that the SV -Iike parainfluenza virus is a more efficient spreader than other viruses causing similar respiratory symptoms, and

that natural outbreaks of kennel cough render the surviving animals uniformly immune to similar parainfluenza infection. Tables IV and V give information collected by the inventor and her colleagues in the study of a spontaneous outbreak of respiratory disease' among laboratory dogs at the University of Pennsylva-' nia. These data reveal a much higher rate of parainfluenza infection than adenovirus infection having similar disease characteristics. Table VI demonstrates resistance to disease on natural challenge of dogs which reperimental challenge with a virulent strain of the virus.

TABLE l Reports of Isolation of an SVS-likc Parainfluenra Virus from Epizootics of Canine Respiratory Disease Population Publication Washington. D C.

Lackland AFB. Texas Ft. Banning. Ga.

New York and New Jersey New Jersey Ithaca. N.Y. Philadelphia. Pa

Quakertown. Pa. Dublin. Va.

laboratory Dogs Binn et al. Proc. Soc. Exp.

Biol. and Mad. lltIl-I. I967 Sentry Dogs (randell et al. An\..l.Vet.Res.

29:2]41. I908 Militar Dogs Binn ct al. AmJ.Vet.Rcs.

Pet Shop and Agppcl and Percy. J. A.V.M.A.

Kennel Dogs [56:1778. I969 Urban Pets Cornell Dog Farm Lie! and Rosenberg. Unpuh d Lief. Unpuhd.

TABLE II Results of Reciprocal Neutralixation Tests on Some Canine Parainfluenva Isolates from Different Sources -Titers with Isolate No.

ATCC VR 666 4 Source Dog No. Serum Penn/343 Penn/h Penn/Hi3 Penn/268 Penn! I'll Binns CQSNBIl U. of P. Vet Hosp. Penn/343 I28 64 (I967) Quakcrtown. Pa. Penn/406 8-H: l6 l6 Kennel (May 1970) U. of P. Lab. (July I970) Penn/I03 l6 l6 l6 (ATCC VR 666) Penn/26K 64 64 I28 (\4 (v4 Penn/I'll I28 I28 I28 64 Walter Reed C 95833 I28 I28 (Binn) convalescent TABLE 111 TABLE VI Antigenic Analysis by H-1 o1- Viruses Recovered from Mongrel Dogs in Virginia and Pennsylvania the La to y p lflot c Results of Natural Challenge of Dogs Recovered from H1 Titers Isolate No. with Penn/343 Scrum Virus Previous No. No. lsolations on Challenge Val/7527 32 Exposure Dogs P1 Adeno Illness Val/7526 32 (a) Va/7522 32 Penn/343 32-64 Para 18 0 8 Penn/103, ATCC VR 666 64 1Q influenza 6 3(5071) 2 Adeno 20 3 6( 30% v 4 0 4( 100" Totals I 24 3(1)) 10(1)) 3 (a)1nc1udcs animals with antil'mdics before start of laboratory epivotic. (h)Thc 13 \irus isolations were made from diflcrcnt dogs. No douhlc infections were encountered TABLE IV Parainfluenva and Adenovirus Infections in an Hpizootic Aniona Laboratory Dogs at the University of Pennsylvania H-1 Virus lsol. Antibody Rises Deaths after Dog No. P1 Adeno P1 Adeno(a) lllness( h) Admission 102 0/64(Cl 0/0 I 103 0164 0/0 t 104 0/0 0/0 115 ()/128 0/0 1 19 0/ 16 8/8 '1- 121 ()[256 16/8 178 0/128 32/ 16 216 0/0 16/8 '1- 224 v ()/ND ()/ND Died in two weeks 268 (1/256 64/32 292 (1/256 8/8 2914' "[128 16/128 1 396 010 16/0 405 (1/64 64/64 41 I ()/(l (1/128(d) 412 ()IND (DIN!) Euthanized one week 413 (I/ND (l/ND F-ver onl Died in one week 423 0/64 0/64 t 424 8/ 16 16/16 426 (1/(] 16/16 Died in five weeks 575 01128 16/8 655 "/8 16/8 667 256/256 16/8 699 256/32 64/8 700 0/0 64/16 701 7 64/64 64/32 81 1'1 0/64 X/ l 28 1: 824 010 512/256 Totals /28 4/28 15/28 3/ 28 /28 4/28 (amgainst virus isolated 1mm N 2V4 (in: mild (111; more severe signs (I510 121 V (d lagainst 1(11 only TABLE V Incidence of Parainfluenm and Adenovirus Infections in an Epivootic of Respiratory Disease Among laboratory Dogs Pre-cxisting No. No: Dogs No Virus No. with Rises No. 111 with Virus Total Virus Antibodies Dogs With Rises 1nso1ations or [solutions 1solations or Antibody Rises Illness Para- None 24 15"" (62%) 15 (6254) 16 (67 1) 14/160179) 14 (5851) influenza a 1:8 4 0 0 0 0 0 Total 28 15 (54%) 15 (549?) 15(5794) 14/16 (87%) 14 Adeno None 9 2"" (22; 3 (33V?) 3 (33% 1 3/3 3 (33% a 1:8 19 1(2'1) 1(2?) 1(24) 111(100'11 1(254) Total 28 3 (101 11 4(1471) 4(141) 4/4(10(YK) 4 (14%) None Identified 28 2 (7'24 ""No comalcsccm scrum became available from a dog which shed \irus and died. However another dog sustained an antibody rise but no virus was isolated from him ""No com'aliiccnt scrum obtained from one dog hich shed virus and (lied.

TABLE Vll Resistance ms riinfiml Challenge with Pcnn/343/67 CanineParainfluengu Virus of Dogs Previously infected in Nature or Experimentally with the Same Virus "Rcspiratory symptoms confirmed by positive radiologic and/or histugunlmlogic findings. ""Prc-existing titers were low enough to show booster l'C$[X |IL$ to viral antigen introduced into nose and throat even without multiplicttion.

SUMMARY OF INVENTION 7 This invention provides a live-virus SV -,-like canine parainfluenza vaccine whichcan be stored for many months at 4C. in its lyophilized form. This vaccine has been found to be safe and immunogenic and capable, under field conditions, of limiting and controlling kennel cough. intramuscular administration is presently preferred. Vaccinees suffer no side effects and rarely if ever excrete virus when this route is used. Furthermore, on giving the vaccine by the natural route (into the nose and throat) although they shed virus for about one week. none of the animals or their contacts become ill. (Table VI") The use of a continuous mammalian kidney cell line, such as Madin Darby Canine Kidney (MDCK) cell cultures, for the original isolation of the strain, for its successive passage and in the manufacture of the vaccine excludes the likelihood of latent viruses being transmitted to the vaccinee through the vaccine. Furthermore, the use of a virus vaccine for immunization of canines, which has been grown in cell cultures derived from the same species, excludes the possibility of the induction of allergic or hypersensitive reactions in the vaccinees. Field tests indicate that the berg FJ. Lief FS, Todd JD, and Rief 15, Studies of Ca- 1 nine Respiratory Viruses. I. Experimental infection of Dogs with an SV -Iike Canine Parainfluenza Agent". AM. J. of Epidemiology 94: 147 (1971 or f s-i i lates obtained fromother ep'iZootics by the inventor 'or other investigatorstAppel, M.,Pickerill;P. H., Menegus, M., Percy D; H; Parsonon; l. Mfiand Shefiy, B. E. Current Status ofCanine- Respiratory Disease. 20th Gaines Vet. Syrnposium, Manhattan. Kansas College Vet.Med.-, K.-S.Q. Newer Knowledge About Dogs. Oct. 22; l970, pp. 1 1543- Awaccine strain at the fifth pas sage level (Penn/ 103/70) has be'e'ndeposited with the American Type Culture Collection (ATCC) which has assigned it the designation ATCC VR'666. During the term of this patent, it may be. obtained from the collec tion by anyone. It was initially isolated in MDCK cells and multiplied to high titers on subpassage in these cells. Vaccine prepared from this strain was found to be safe and caused resistance to natural challenge under field conditions. I

The canine parainfluenza virus is cultivated for the vaccine by successively passing the original isolate in MDCK cells employing as seed. infected supernatant fluids harvested from the previous passage.-The inoculated cultures are incubated at each passage in the stationary position for a period of 6 to 7 days at a temperature of 36.

Propagation of the MDCK cells may be carried out by any of the standard methods described in the literature. A tissue culture system comprising Eagles minimal essential medium in Earlle'sbuffered salt solution. with the addition of 10% fetal calf serum, I00 units of penicillin and lOO mcg of streptomycin per ml is one of the recommended standard techniques for growing MDCK cultures. For rnaintenance of the cultures, 2% calf serum is preferred. Before inoculation with virus the MDCK cell layers should be washed with Medium I99 supplemented with the above antibiotics. Following inoculation with virus, the cultures should be maintained in the same Medium with the addition of stabilizers as 0. l X SP0 and 0.5% gelatin. it is to be understood that in referring to physiologic solutions, the letter X is known to refer to the concentration of the physiologic solution in question. It is common for physiologic solutions of the general type of SPG to be supplied in the lOX form, meaning that the solution is to be diluted to one-tenth of its strength. of LX, in order to provide a solution having constituents. each of which is present in the. desired balanced amounts.

For preparatioh of the vaccine, virus in the supematant to which the stabilizers have been added lX SP6 and 0.5% gelatin) as well as intracellular virus released through a series of rapid freeze-thaw cycles is used. The resulting suspension is centrifuged at 3000 RPM for 10 minutes to remove cellular debris. This resultant clarified supernatant represents the final vaccine product which must be storedat to -C. until needed. This final product usually has an HA titer of l :l28 to 1:256 when tested with guinea pig erythrocytes and a TCD of i0""" per milliliter when titrated in MDCK period of time at refrigerator temperatures. Prepara- (ton of this lyophilizcd product entails one less freezethaw cycle prior to clarification than for the aqueous product. On resuspension in the appropriate diluent. such as water (pH 7.0). the lyophilized product was found to have HA and infectivity titers commensurate with that observed for the aqueous product.

TABLE vi'n"- tles incubated in the stationary position require rocking at 15 minute intervals. At the end of the hour, Medium 199 containing antibiotics and stabilizers is added to the inoculated cultures in volumes appropriate for the particular container. The inoculated cultures are incubated for 6 to 7 days. The pH is monitored and excessive acidity controlled by the addition of a small ResulLs in Dogs Given Pl I03 Vaccine" by the Naso-Pharyngeal Route and in Dogs Exposed to Them (isolated from inoculated and contact dogs) "\'uccine contained TCD per ml. *l-m-cr or runny nose or conjunctivitis or sptmtaneous cough.

Began 2 days after inoculation or exposure to inoculated dogs and lasted'alxmt l week "PlD=Post-lnoculation Du "'Serum Neulmliwing Antibod "liter.

PREFERRED EMBODIMENT 1 Preparation of the final aqueous product is'accomplished in two phases. First aseed virus for the vaccine is prepared by subpassing the isolate in MDCK cells four times using an incubation period of 6 to 7 days at 36, a period which was found tofproduce the peak infectivity titers. To each harvest and espccially'to'that of the passage to be used as seed virus for'vaccin preparation stabilizers are added before each is divided into small aliquots and frozen at less than -'-"70 C. until needed. me stabilizer. SP6. is added to give a final concentration of IX according to the method described by Bovamick. et al, The influence of Certain Salts, Amino Acids, Sugars and Proteins on a- Stability of Rickettsiae", J. Bact. 89:509-522 (1950) and sterile gelatin is added to give a final concentration of 0.5%. For convenience, harvests of initial serial passages may be frozen and repassed at a later time to form additional seed virus stocks for vaccine production. These seed stocks. which represent supernatant fluids'from infected cells, usually have a hemagglutination- (HA) titer with guinea pig red cells of 1:64 and'an infectivity titer (TCD of 10".:7 per milliliter. I

In the second phase of vaccine production. virus is propagated in MDCK cells grown in 32 ounce bottles or in roller tubes usingaseed virus which hasunde'rgoneat least4- 5 passages. Before inoculation .with the virus. the growth medium is removed and-cells are washed twice with Medium 199 containing 100 u peniamount of fresh medium to maintain a p| -l between 7 and 7.2. Such an addition is rarely required.

At the conclusion or this incubation. sterile gelatin to give a final concentration of 0.5% and SP6 to give a final concentration of LOX is introduced into each vessel. The cultures are thenirnrnediately irozen at 70 to 85C. and, again for the sake of convenience. may be storedat temperature until the final vaccine product is v: i

The-final vaccineis produced by subjecting the frozen cultures to atotal of l to freeze-thaw cycles in order to release intracellular virus. The resulting fluid cell suspensions from each container may be and the pools then should be centrifuged at 3,000 rpm for ten minutes. The clarified supernatantfluid represents the final aqueous product. It should be immedicillin and 50 ug streptomycin per milliliter. A volume of 10 dilution ofsced virus in Medium 199 containing the antibiotics and 0. l X SP0 and 0.5% gelatin is introduced into the bottles or tubes in sufficient quantities to cover the cell monolayers. The cultures are then returned to the incubator for one-hour. Boiler tubes returned to the roller drum need no later rocking but hotately distributed in desired volumes in appropriately vials and immediately stored at to C. The final product will have an l-lA titer. of 1:128 to l:256 when tested guinea pigerythrocytes. and a TCD of 10- per milliliter whentitrated in MDCK cell cultures a 7 I I v Because of the relative instability of' the vaccine when stored at temperatures higher than -70C., a

method. of lyophilization was developed to provide a practical means'of preserving the vaccine, TablelX demonstrates-1hr; instahility .;of the aqueous product whenstoredat -2()C.; the temperature of an ordinary freezer. Lyophiiization appears to; have no adverseaffeet on infectivity. Tests indicate that the lyophilized product can be stored at least eight months'at 4 without any infectivity losses. See.Table X. After resuspension; the infectivity stability is the same as that of the aqueous vaccine. See Table Xi. Preparation of the lyophilized product utilizes a single cycle of freezing and thawing of cells and fluid prior to centrifugation. cittrifugation and clarification are accomplished as before and the resulting supernatant then should be immediately lyophilized in 2 to 10 ml. volumes. The lyophilized product may be stored at 4 until needed. The resuspended product should be used immediately.

TABLE IX Stability of ATCC VR 666 Vaccine Stored at Different Temperatures Temp. of Length of TCDSO I og 10 Storage Storage per 0.2m! Change -75C 5 days 0 7 months l0" O 2()C 1 month My -l.5 2% months 7 l0 2.5 4 months l0 l0" -3 to 7 months l0" 3 to -7 7 months IO 10" 'i to -7 TABLE X Stability of Lyophilized ATCC VR 666 Vaccine at 4C 'rcoso Vaccine Storage Timc per ml Change Original Liquid 0 10' 0 A Lyophil kept at 4C l week I()"' 0 1 month H (l 5 months I()' U 8 months It) (I TABLE XI Instability of Lyophilized Vaccine After Resuspension and Storage at-4C.

These changes are the same as occur with fresh harvests of this virus stored at 4(.

Laboratories Research Animal Facilities a large kennel which specilizes in supplying conditioned m ongrel dogs for research purposes. This kennel had experited. It was estimated that 10% of the total population died weekly because of respiratory disease. These deaths meant that more than 50% of the dogs admitted weekly would not survive their six to eight week stay.

In the first trial, two groups of newly admitted mongrel dogs comprising 31 animals were segregated from the rest of the population and given the aqueous vaccine which had been stored at C. and contained 10 TCD per milliliter. The dogs were administered one milliliter doses in both the right and left neck muscles. Seven to ten days after vaccination all but four of the dogs were introduced into the general population. Naso-Pharyngeal swabs for virus isolation were taken on days 0, 3, 7, l0 and 20. Blood samples for antibody assays were taken on the day of administration and four weeks after vaccination. All animals were followed for at least six weeks and the four which remained in isolation were retained for 15 months in order to study antibody persistence.

.All of the animals remained well even in the face of natural challenge. None shed virus accept for one, from which virus was recovered on the seventh day only. See Table XII. Although no dogs had had detectable circulating antibodies on admission to the study, four weeks after vaccination 29 of the 3 1 showed sew-conversion. with a mean I-I-I titer of 1:55. Neutralizing antibodies which appear later than H-] antibodies had .a mean of 1:78 at this time. See Table XIII. Neutralizing antibodies developed and persisted at: high titers in all four dogs kept for 15 months. Their H-I antibodies. on the other hand. although still present at l5 months. dcclined 2 to 4 fold from the one month level. See Table XIV.

Example 2 Using the same facilities as in Example l. a total of 38 dogs were, inoculated with a vaccine which contained l0' TCD; per milliliter. Twenty of those dogs were inoculated intramuscularly and the remaining 18 dogs were inoculated subcutaneously in the neck region. The rate and level of sero-conversion for both routes were about the same as found in the first trial (Table XV). Thus. it appears that the vaccine is equally capable of stimulating circulating antibodies when given intramuscularly or subcutaneously.

TABLE XII Illness and Virus Excretion in Dogs Given ATCC VR 666 Vaccine Stored Frozen at -75C Dose and No. No. No. Excreting Virus on Total No.

Route Dogs Ill Days After Vaccination Excreting Day 0 Day 3 Day 7 Day II) Day 20 2' X IO TCDSO 3| 0 (l (J l O (J 1/} l/ (37:)

intram uscularly In this trial, an attempt was made to correlate infectivity losses due to high storage temperatures with immunogenicity. Nine dogs were inoculated intramuscularly with aqueous vaccines stored at 75C., nine with the samevaccine lot stored at "C. and 10 with the same vaccine lot stored at 4C. Table XVI shows there is a direct correlation between the level of antibody response and the amount of live virus remaining in the vaccine. The highest titers were obtained with the 75C. stored preparation and the lowest with the 4C. stored material.

A similarassay was carried out with lyophilized vaccine, to compare its immunogenic'ity with that of aqueous vaccines properly stored at 75C. Twenty-nine dogs were given 2 milliliters intramuscularly of a resuspended preparation containing 10 TCD per milli- TABLE Xlll Antibody Responses of Dogs to ATCC VR 666 Vaccine Stored Froyen at C Dose and No. H-l Titers Neutrnliving Titers Route Dogs No. Converting Median Menn No. Converting Median Mean 2X ll) TCDSO ll 29/3] (93%) X/64"" 8/55 25/29 (869;) 8/64 X/7 intrumusculairly ""Nunierntor prevaccinntion and denominator postvztccinntion titers ""Ol' positive responders ""l'wo dogs not tested TABLE XIV Persistence of Antibodies in Dogs Given ATCC VR (s66 Vaccine. Stored Frozen at -75( Dose and Dog H-l Titers Neut. Titers Route N0. 1 1 2 a 9 l5 0 l 2 a 9 l5 day mo mos mos mos mos day mo. mos. mos. mos. mos.

2X l(l 4655 3 I28 64 32 32 32 8 :50 I28 156 256 I28 TCDSU 4662 8 I28 64 32 64 64 8 32 I28 256 128 256 intra 4679 8 64 32 32 32 8 32 e4 64 64 muscularly 46x4 8 64 32 32 64 4 8 I 6 I6 I28 I28 I28 TABLE XV H-l Antibody Responses of Dogs to ATCC VR 666 Vaccine Stored Frozen at 75C and Given by Difl'erent Routes PreTiters Post-Titers No. Showing Dose and Route Total No. Dogs Sero conversion G.M. Range G.M.* Range 2 X l()' TCDSO 20 I7 8 8 64 l6-Sl2 intramuscularly 2 X l0" TCDSO l8 l7 (9471) 8 8 5l 8-512 subcutaneously of positive res onders Example 3 40 liter, and it became clear that the antibody responses were very similar to that induced by vaccines stored at 75C. See Table XVll.

Example 4 Beginning in the middle of February I970, all new arrivals to the mongrel dog colony were inoculated intramuscularly as described above with 2 milliliters of the ATCC VR 666 vaccine which was kept at -75C. Since it was known that antibodies to this virus could be detected as early as the eighth day after exposure. the newly vaccinated dogs were kept apart for seven to 10 days before being introduced into the general population. This policy was followed until almost lOO% of the population represented vaccinated animals. Thereafter, animals were introduced into the general population without the 7 to ID day segregation period.

TABLE XVI l Antibody Responses of Dogs to ATCC VR 666 Vaccine Stored at Different Temperatures for Five Months TC D50 Numemtor prcvuecination and denominator postvuccinntion titers Antibody Responses of Dogs to ATCC VR 666 Vaccine Lyophilized Dose and No. H-I Titers Neutralizing Titers Route Dogs No. converting Median Mean No. convening Median Mean 2 X TCDSO intramuscularly 29 26 (90%) 8/64"" 8/48 (707c)" 8/l6 8/83 ""Nurnerator=prevaccination and denominator=postvaccination titers 01' positive responders "H-l antibodies ap ear before neutralizing antibodies. These sera were taken before neutralizing antibodies reach their peak.

As can be seen from Table XVIII, mortality rates for the five month period preceding the inception of the vaccination program revealed that when computed on a four week basis an average of 5.5 10.8% of the resident population succumbed each week to respiratory disease. As would be normal in a changing population of this kind there was considerable variation in the weekly death rate. Some weeks it reached as high as 14-16% after which it fell, probably due to the reduction of the number of susceptibles remaining. With the introduction of new susceptibles, this reduction was always followed by a marked increase in the weekly death rate.

The impact of the vaccinations did not become apparent until a majority of the dogs in the colony were vaccinated. In the week ending Mar. 24, 1971, 50% of the resident dogs consisted of vaccinated animals. The mortality rate per total population that week was 4%. As the percentage of vaccinated dogs increased the mortality rates continued to fall. By the time 70-100% of the dogs in the kennel were vaccinated, the weekly death rate was 2%. From Apr. 5 to May 8, 1971 no vaccine was available, and new unvaccinated dogs were admitted while vaccinated dogs were sold. During this period the mortality rates rose to 4.7% from a figure of 2.1% recorded the week before. Reinstitution of vaccinations immediately caused a fall in the death rate. Before the old rate of approximately 2% was reestablished, aqueous vaccine which had been stored at -20C. was inadvertently administered. This error was discovered when it was noticed that more dogs were dying of respiratory disease and indeed there was an increase in the average weekly mortality rate. Resumption of inoculation with potent vaccine gradually forced the mortality rate to a low of between 0.4 and 1% which has persisted.

For an assessment of the overall mortality rates during the control and vaccinated periods see Tables XIX and XX. It can be seen that the weekly death rate for the control period was 7.4%. Even including the period when no vaccine was available the vaccination period death rate was 3.92%. Counting only that period when vaccine was available the death rate became 3.39%. Applying the Chi test, p was less than 0.001. Table XXI illustrates the fact that these rates could be maintained with the use of lyophilized vaccine.

The utility of this vaccine in the prevention of kennel cough is best revealed by examining the death rates of dogs admitted to the kennel beforeand after inception of the vaccination with the ATCC VR 666 vaccine (See Tables XXII and XXIII). Of 1.980 dogs admitted from September to March 1971, 1,141 of these dogs died or were euthanized because of severe respiratory disease. After vaccination was instituted only 626 of the 2.391 dogs admitted during the next 9 /2 months died or where euthanized for the same reason. Instead of a death rate of 58%. during the vaccination period only 26% of the dogs succumbed to respiratory disease. In 1972 this death rate decreased even more, resulting in the survival of 79 dogs out of every 100 admitted, representing adecrease in mortality rate of 64%.

The loss of 20% of all incoming dogs to respiratory disease is still a problem. A number of other agents can mimic the symptoms of kennel cough. To some extent, this death rate may be explained by the admission of some animals which arrive preinfected, not only with canine parainfluenza virus butmost likely with canine distemper, ICH and [CH-like viruses and probably bacterial agents as well. No vaccine can prevent these diseases from running their full course. However. the use of this canine parainfluenza virus vaccine in all incoming dogs clearly prevented the transmission of a major Weekly Mortality Rates in a Mongrel Dog Kennel During Control and Vaccinated Periods Using Frozen ATCC VR 666 a No. Dead or Vaccine Average Weekly Mortality Interval Total Dogs at Risk Euth. for Illness Status per Total Population 9/16-10/13/70 2894 313 0 10.8 10/14-11/11/70 3015 203 0 6.7 12/10-1/6/71 2328 196 (l 8.3 1/7 2/3/7l 2975 I62 0 5.5 2/4 2/l7/7l 1245 101 0 8.1

Began vaccinations week of 2/10; 22 of 601 dogs vaccinated Z/10-3/10/71 2268 166 10-40'/1 7.3 3/1 l- 4/7/7l 2074 gradually increased 2 l to 4/8 5/5/71 2676 no vaccine. gradually 4.7

reduced to 40% 5/6 6/2/7l 2930 I33 404K171 4.5 6/3 6/30/71 242) 166 20 C vaccine with low titer used in this period 6.) 7/1 7/28/71 2492 R7 1009 3.4 7/29-8/25/71 25X) 31 10(1'71 8/26-9/22/71 2278 25 100% 1.1 9/23-10/6/71 104) 4 10(171 4 cause of respiratory infections to other residents in the ("Rum Deaths kennel. There is no doubt too that this'ca'nine parain- Dog wccks of Rlsk fluenza ivirus vacclne when p'rePared and adrplnlsFered ""lncludes 4/7 to 5/l9 when no vaccine was uwd and 7% of dogs as described is safe, immunogenic and efficacious in rein population vaccinated fell to 45 and 5/2 to 6/30 when low ducing mortalitics associated with respirutoryl'disease S titer vaccine stored at 20 was used. Durmg these periods some death increases were noted. outbreaks occurring in kennels. (mp 00] TABLE XlX Average Weekly Mortality Rates per Total Population for Nun-Vaccinated and Vaccinated Periods UsingFroren ATCC VR 666 ""Total number dogs survived weekly "Total number dogs at risk weekly Deaths Total Dog Wecksof Risk TABLEXXI Weekly Mortality Rates After Beginning Vaccination with Lyophilized Preparation Wcelt Total Population No. Dogs No. Dead or 7: Mortality per Admitted Euthi for Illness Total Population 2/l5-2/2l/73 222 40 4 1.8 2/22 2/28/73 243 48 3 L2 2/29-3/7/73 253 45 8 3. 3/8- -3/l4/73 255 44 0 O 3/l5-3/ 2l/73 273 47 2 0.8

Total I246 224 17 l .3

Beginning 2/!2/73 new dogs were given 2 X 10"" TCDSU intramuscularly of this preparation.

TABLE XX Average Weekly Mortality Rates per total Population for Non-Vaccinated and Vaccinated Periods Using Frozen ATCC VR 666 Time Period Vaccine Died Survived Total" Rate 9/l6/70-2/l l/7l No 885 10,961 I L846 7.47% 2/ l 0/7 ll 0/6/7 l Yes 792 l9 434 20,226 3.92%"

""Total number dogs survived weekly ""Total number of dogs at risk weekly TABLE XXII Influence of Use of ATCC VR 666 Vaccine on the Mortality Rates per Dogs Admitted to the Mongrel Dog Colony No. Dogs Vaccine No. Dead or Mortality Time Period Admitted Status Euthanized Rate (7:)

9/l6/70 3/l()/7l 1980 None to 2/lO/7l 1141 57.6

l 0- 40% vaccinated 2/ 10-3/ l O sumcd hut 2()C vaccine mainly used 7/l/7l-l0/6/7l 808 I007: vaccinated I47 li'Ll TABLE XXlll Mortality Rates in Dogs lntroduced into the Mongrel Dog Kennel During Control and Vaccinated Periods NOTE; (ontrol period includes 2/ll) JIM/7| when less than 4071 of dogs were vaccinated. After vacc zition with Pl I03 i'istitutcd there were periods of time when no vaccine was available or \accin rated by storage at C was inadvertently used. During lhcsc periodsv grdlcss of the time of thc your. morbidity and mortality rates rose. hut tcll again as soon as vaccine with proper inl'cclivity was reintroduced. (Table l7).

Example 5 This test was performed in another Flow Laboratories Research Animal Facilities kennel which was used for breeding and raising beagles. The total population of this kennel was about 500 dogs. Severe respiratory disease symptoms appeared chiefly in young puppies five to eight weeks old. Better than 50% of the young animals contracted the disease, and most of those who did contract the disease died. As in the first population. a parainfluenza agent similar to the proposed vaccine strain was recovered from these sick puppies, and a plan for inoculation of the puppies was formulated.

Due to a decision by Flow Laboratories to disband the beagle population within two months, the testing possibilities for the vaccine were severely curtailed. Between Feb. l9, and Apr. 10, 1971, 230 puppies ranging in age from five to seven weeks were vaccinated. They were given 1 milliliter of the vaccine divided equally and injected into the neck muscles. Ordinary mortality rates due to respiratory disease in this age group suggested that 5060% of the dogs would have contracted the disease and died had they not been vaccinated. in the vaccinated group the mortality rate was The shortened testing period considerably reduced the reliability of this test. Parainfluenza virus was isolated from some of the vaccinated dogs which died as well as from other nonvaccinated young dogs which were sick or dying from respiratory disease. Many of the vaccinated puppies which died may have succumbed to natural infection prior to vaccination. Although more extensive and better controlled studies are needed, preliminary data demonstrates that the canine parainfluenza tracheobronchitis vaccine shows practical utility in populations of this kind.

I claim:

1. A method of producing an aqueous canine tracheobronchitis vaccine comprising the step of serially passaging a SV -like canine tracheobronchitis virus strain in a continuous-line mammalian kidney cell culture free of extraneous agents to produce said aqueous canine tracheobronchitis vaccine.

2. A method in accordance with claim 1, comprising the additional step of first isolating said virus strain in said cell culture.

3. A method in accordance with claim 1, comprising the additional steps of increasing the virus yield by releasing the intracellular virus through rapid freeze-thaw 18 cycles of said cell culture and centrifuging the resulting fluid-cell suspension at low speed to produce a Clarified supernatant aqueous vaccine.

4. A method in accordance with claim 1, comprising (a) the additional steps of adding SPG to give a final concentration at lX SPG and gelatin to give a final concentration at 0.5% gelatin to stabilize the HA and infectivity titers of the harvests of virus from said viruscontaining cell culture; and (b) adding to the medium during propagation the virus-containing cell culture SPG to give a final concentration of 0. 1X SPG and gelatin to give a final concentration of 0.5% gelatin to insure against inactivation during propagation.

5. A method in accordance with claim 1, wherein said virus strain is serially passaged at least five times in Madin Darby Canine Kidney Cells.

6. A method in accordance with claim 1, wherein said virus strain is Penn/lO3/70, ATCC VR 666.

7. A method in accordance with claim 5, wherein each passage is incubated at 36C. for 6 to 7 days.

8. A method in accordance with claim 3, wherein one to two freeze-thaw cycles are used to release the virus particles from the cells.

9. A method of producing an aqueous canine tracheobronchitis vaccine comprising the steps of:

l. first isolating and then serially passaging the canine tracheobronchitis virus strain designated ATCC VR 666 at least five times in Madin Darby Canine Kidney cell culture. each passage being incubated at 36C. for 6 to 7 days;

. stabilizing the HA titer and infectivity titer by adding SPG to a final concentration of IX SPG and gelatin to a final concentration of 0.5% gelatin for harvest of the virus strain and SPG to a final con centration of 0.1X SPG and gelatin to a final concentration of 0.5% gelatin for propagation of the virus strain;

3. increasing the virus yield by releasing the intracellular virus through one to two rapid freeze-thaw cycles of said cell culture; and

4. centrifuging the resulting fluid-cell suspension at low speed to produce a clarified supernatant aqueous vaccine.

10. A method of producing a lyophilized canine tracheobronchitis vaccine comprising the steps of:

l. serially passaging an SV -,-like canine trachcobronchitis virus strain in a continuous-line mammalian kidney cell culture free of extraneous agents;

2. at harvest of the virus strain from said cell culture, adding SPG to a final concentration of IX SPG and gelatin to a final concentration of 0.5% gelatin, and during propagating of the virus strain adding SPG to a final concentration ofO. 1X SPG and gelatin to a final concentration of 0.5% gelatin;

3. increasing the virus yield by releasing the intracellular virus through rapid freezcthaw cycles of said cell culture;

4. centrifuging the resulting fluid cell suspension to produce a clarified virus containing supernatant;

5. lyophilizing said supernatant;

6. storing the lyophilized product at 4C. until needed for use; and

7. resuspending said product in an appropriate resuspending medium to obtain said lyophilized vaccine.

11. A method in accordance with claim 10, comprising the additional step of first isolating said virus strain in said continuous-line mammalian kidney cell culture.

12. A method in accordance with claim 11, wherein said virus strain is first isolated and then serially passaged at least five times in Madin Darby Canine Kidney cell culture.

13. .Amethod in accordance with claim 10, wherein said virus strain is Penn/lO3/70 ATCC VR 666.

l4..A method in accordance with claim 12, wherein each passage is incubated at 36C. for 6 to 7 days.

15. A method in accordance with claim 10, wherein a single freeze-thaw cycle is used to release the virus strain from the cell culture.

16. A method of producing a lyophilized canine tracheobronchitis vaccine comprising the steps of:

l. first isolating and then serially passaging the canine tracheobronchitis virus strain designated ATCC VR 666, at least five times in Madin Darby Canine Kidney Cell culture, each passage being incubated at 36C. for 6 to 7 days;

2. at harvest of the virus strain from said cell culture, adding SPG to a final concentration of IX SPG and gelatin to a final concentration of 0.5% gelatin, and during propagating of the virus strain adding SPG to a final concentration of O. l X SPG and gelatin to a final concentration of 0.5% gelatin;

3. increasing the virus yield by releasing the intracellular virus through a rapid freeze-thaw cycle of said cell culture;

4. centrifuging the resulting fluid-cell suspension to produce a clarified virus-containing supernatant;

5. lyophilizing said supernatant;

6. storing that lyophilizing product at 4C. until needed for use; and

7. resuspending said product in an appropriate resuspending medium to obtain said lyophilized vaccine.

17. A method of inducing immunity to canine tracheobronchitis comprising parenterally administering a canine tracheobronchitis vaccine prepared as in claim 1 in sufficient quantities to produce antibodies without causing undesired symptoms of the disease.

18. A method in accordance with claim 17, wherein the vaccine is administered intramuscularly.

19. A method in accordance with claim 17, wherein dosage given of vaccine with a TCD of IO per milliliter is approximately 2 milliliters per adult animal and 1 milliliter per puppy.

20. A method of inducing immunity to canine tracheobronchitis comprising parenterally administering a canine tracheobronchitis vaccine prepared as in claim 10, in sufficient quantities to produce antibodies without causing undesired symptoms of the disease.

21. A method in accordance with claim 20, wherein the vaccine is administered intramuscularly.

22. A method in accordance with claim 20, wherein the dosage given a vaccine with a TCD of l0 per milliliter of approximately 2 milliliters per adult animal and l milliliter per puppy.

23. A method of stabilizing the HA titer and infectivity titer of a virus-containing cell culture comprising the step of adding SPG and gelatin to stabilize said HA titer and said infectivity titer.

24. A method in accordance with claim 23, wherein SPG is added to give a final concentration of IX SPG and gelatin is added to give a final concentration of 0.5% gelatin for harvest of the virus from said viruscontaining cell cultures, and SPG is added to give a final concentration of 0.1X SPG and gelatin is added to give a final concentration of 0.5% gelatin for propagation of said virus-containing cell culture.

25. A method for preserving the infectivity of a virus harvest during lyophilization comprising -the step of adding SPG to give a final concentration of IX SPG and gelatin to give a final concentration of 0.5% gelatin. 

1. A METHOD OF PRODUCING AN AQUEOUS CANINE TRACHEOBRONCHITIS VACCINE COMPRISING THE STEPS OF SERIALLY PASSAGING A V5LIKE CANINE TRACHEOBRONCHITIS VIRUS STRAIN IN A CONTINUOUS-LINE MAMMALIAN KIDNEY CELL CULTURE FREE OF EXTRANEOUS AGENTS TO PRODUCE SAID AQUEOUS CANINE TRACHEOBRONCHITIS VACCINE.
 2. A method in accordance with claim 1, comprising the additional step of first isolating said virus strain in said cell culture.
 2. at harvest of the virus strain from said cell culture, adding SPG to a final concentration of 1X SPG and gelatin to a final concentration of 0.5% gelatin, and during propagating of the virus strain adding SPG to a final concentration of 0.1X SPG and gelatin to a final concentration of 0.5% gelatin;
 2. at harvest of the virus strain from said cell culture, adding SPG to a final concentration of 1X SPG and gelatin to a final concentration of 0.5% gelatin, and during propagating of the virus strain adding SPG to a final concentration of 0.1X SPG and gelatin to a final concentration of 0.5% gelatin;
 2. stabilizing the HA titer and infectivity titer by adding SPG to a final concentration of 1X SPG and gelatin to a final concentration of 0.5% gelatin for harvest of the virus strain and SPG to a final concentration of 0.1X SPG and gelatin to a final concentration of 0.5% gelatin for propagation of the virus strain;
 3. increasing the virus yield by releasing the intracellular virus through one to two rapid freeze-thaw cycles of said cell culture; and
 3. increasing the virus yield by releasing the intracellular virus through rapid freeze-thaw cycles of said cell culture;
 3. A method in accordance with claim 1, comprising the additional steps of increasing the virus yield by releasing the intracellular virus through rapid freeze-thaw cycles of said cell culture and centrifuging the resulting fluid-cell suspension at low speed to produce a clarified supernatAnt aqueous vaccine.
 3. increasing the virus yield by releasing the intracellular virus through a rapid freeze-thaw cycle of said cell culture;
 4. A method in accordance with claim 1, comprising (a) the additional steps of adding SPG to give a final concentration at 1X SPG and gelatin to give a final concentration at 0.5% gelatin to stabilize the HA and infectivity titers of the harvests of virus from said virus-containing cell culture; and (b) adding to the medium during propagation the virus-containing cell culture SPG to give a final concentration of 0.1X SPG and gelatin to give a final concentration of 0.5% gelatin to insure against inactivation during propagation.
 4. centrifuging the resulting fluid-cell suspension at low speed to produce a clarified supernatant aqueous vaccine.
 4. centrifuging the resulting fluid cell suspension to produce a clarified virus containing supernatant;
 4. centrifuging the resulting fluid-cell suspension to produce a clarified virus-containing supernatant;
 5. lyophilizing said supernatant;
 5. lyophilizing said supernatant;
 5. A method in accordance with claim 1, wherein said virus strain is serially passaged at least five times in Madin Darby Canine Kidney Cells.
 6. A method in accordance with claim 1, wherein said virus strain is Penn/103/70, ATCC VR
 666. 6. storing the lyophilized product at 4*C. until needed for use; and
 6. storing that lyophilizing product at 4*C. until needed for use; and
 7. resuspending said product in an appropriate resuspending medium to obtain said lyophilized vaccine.
 7. resuspending said product in an appropriate resuspending medium to obtain said lyophilized vaccine.
 7. A method in accordance with claim 5, wherein each passage is incubated at 36*C. for 6 to 7 days.
 8. A method in accordance with claim 3, wherein one to two freeze-thaw cycles are used to release the virus particles from the cells.
 9. A method of producing an aqueous canine tracheobronchitis vaccine comprising the steps of:
 10. A method of producing a lyophilized canine tracheobronchitis vaccine comprising the steps of:
 11. A method in accordance with claim 10, comprising the additional step of first isolating said virus strain in said continuous-line mammalian kidney cell culture.
 12. A method in accordance with claim 11, wherein said virus strain is first isolated and then serially passaged at least five times in Madin Darby Canine Kidney cell culture.
 13. A method in accordance with claim 10, wherein said virus strain is Penn/103/70 ATCC VR
 666. 14. A method in accordance with claim 12, wherein each passage is incubated at 36*C. for 6 to 7 days.
 15. A method in accordance with claim 10, wherein a single freeze-thaw cycle is used to release the virus strain from the cell culture.
 16. A method of producing a lyophilized canine tracheobronchiTis vaccine comprising the steps of:
 17. A method of inducing immunity to canine tracheobronchitis comprising parenterally administering a canine tracheobronchitis vaccine prepared as in claim 1 in sufficient quantities to produce antibodies without causing undesired symptoms of the disease.
 18. A method in accordance with claim 17, wherein the vaccine is administered intramuscularly.
 19. A method in accordance with claim 17, wherein dosage given of vaccine with a TCD50 of 106.7 7.7 per milliliter is approximately 2 milliliters per adult animal and 1 milliliter per puppy.
 20. A method of inducing immunity to canine tracheobronchitis comprising parenterally administering a canine tracheobronchitis vaccine prepared as in claim 10, in sufficient quantities to produce antibodies without causing undesired symptoms of the disease.
 21. A method in accordance with claim 20, wherein the vaccine is administered intramuscularly.
 22. A method in accordance with claim 20, wherein the dosage given a vaccine with a TCD50 of 106.7 7.7 per milliliter of approximately 2 milliliters per adult animal and 1 milliliter per puppy.
 23. A method of stabilizing the HA titer and infectivity titer of a virus-containing cell culture comprising the step of adding SPG and gelatin to stabilize said HA titer and said infectivity titer.
 24. A method in accordance with claim 23, wherein SPG is added to give a final concentration of 1X SPG and gelatin is added to give a final concentration of 0.5% gelatin for harvest of the virus from said virus-containing cell cultures, and SPG is added to give a final concentration of 0.1X SPG and gelatin is added to give a final concentration of 0.5% gelatin for propagation of said virus-containing cell culture.
 25. A method for preserving the infectivity of a virus harvest during lyophilization comprising the step of adding SPG to give a final concentration of 1X SPG and gelatin to give a final concentration of 0.5% gelatin. 